Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Role of mast cells in zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice.

Zymosan-induced peritonitis was investigated in mast cell-deficient WBB6F1 mice and in Balb/c mice pretreated with mast cell stabilizer (cromolyn) or antagonists of histamine receptors (mepyramine, triprolidine, cimetidine, or ranitidine). The inherited mast cell deficiency in W/Wv knockouts of WBB6F1 mice impaired significantly the level of histamine and plasma exudation (measured 30 min after stimulation) as well as the influx of exudatory leukocytes, accumulation of plasma and exudate chemoattractants, and the release of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) measured at 6 h of inflammation. All of those factors were fully restored after selective intraperitoneal reconstitution of W/Wv mice with bone marrow-derived mast cells from their control +/+ counterparts. Cromolyn pretreatment of Balb/c mice reduced exclusively the early plasma exudation and histamine influx. Blocking of histamine receptors inhibited not only the early plasma exudation but also temporarily diminished primary leukocyte influx and levels of MCP-1 and IL-1beta. In conclusion, mast cells play an important role in the initiation of zymosan-induced peritonitis and modulate its further course.

Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice.

Peritoneal inflammation is a convenient model for comparisons of modulatory effects of morphine in phylogenetically distant vertebrates. Both in salmon and mice morphine injected intraperitoneally together with an irritant (thioglycollate) significantly inhibits inflammation as estimated by the number of peritoneal leukocytes. The low number of exudate cells in morphine-treated animals seems to be compensated by their high activity, as evidenced by the enhanced phorbol myristate acetate-induced respiratory burst. The morphine-inhibited influx of leukocytes into the irritated peritoneal cavity correlates with the morphine-lowered level of plasma chemotactic factors both in fish and mice. It implies that morphine impairs the level of plasma chemotactic factor either directly (affecting their release from the resident peritoneal cells) or indirectly (decreasing the number of inflammatory leukocytes by inhibition of their migration from hemopoietic sites). The inhibitory effects of morphine on both the cell number and chemoattractant level are completely reversed by the naltrexone pretreatment, which implicates the involvement of opioid receptors.

Immunomodulatory effect of riboflavin deficiency and enrichment - reversible pathological response versus silencing of inflammatory activation.

Ariboflavinosis, that is, vitamin B2 deficiency, is a common problem affecting the populations of both developing and affluent countries. Teenagers, elderly people, pregnant women, and alcohol abusers represent groups that are particularly susceptible to this condition. This study was aimed to determine the effect of different riboflavin concentrations (deficiency and supplementation) on macrophages response induced by bacteria or yeast-derived factors i.e. lipopolysaccharide (LPS) and zymosan, respectively. Mouse macrophage RAW 264.7 cells were cultured for 5 days in a medium with a riboflavin concentration corresponding to moderate riboflavin deficiency (3.1 nM), physiological state (10.4 nM), or vitamin pill supplementation (300 nM). On the third or fourth day of deprivation, the medium in some groups was supplemented with riboflavin (300 nM). Macrophages activation were assessed after LPS or zymosan stimulation. Short-term (5 days) riboflavin deprivation resulted in the pathological macrophages activation, manifested especially in a reduction of cell viability and excess release of tumor necrosis factor-α (TNF-α) and high-mobility group box 1 (HMGB1) protein. Moreover, the levels of inducible nitric oxide synthase (iNOS), nitric oxide (NO), heat shock protein (Hsp72), interleukin 1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), and interleukin 10 (IL-10) decreased after riboflavin deprivation, but medium enrichment with riboflavin (300 nM) on the third or fourth day reversed this effect. In the riboflavin-supplemented group, LPS-stimulated macrophages showed lower mortality accompanied by higher Hsp72 expression, reduction of Toll-like receptor 4 (TLR4) and TNF-α, and elevation of NO, IL-6, and IL-10. Moreover, the TLR6, NO, iNOS, IL-1ß, MCP-1, and the keratinocyte chemoattractant (KC) levels significantly decreased in the zymosan-stimulated groups maintained in riboflavin-enriched medium. We conclude that short-term riboflavin deficiency significantly impairs the ability of macrophages to induce proper immune response, while riboflavin enrichment decreases the proinflammatory activation of macrophages.

The effect of laboratory environment on the morphology of the spleen and the thymus in the yellow-bellied toad, Bombina variegata (L.).

The morphological changes were observed in the spleen and the thymus of the yellow-bellied toad, Bombina variegata, kept under standard laboratory conditions. The mean splenic weights of toads studied soon after capture in July and September were 16.0 mg and 17.7 mg, respectively. In contrast, the mean splenic weights significantly decreased in animals maintained in the laboratory from July till September (to 11.6 mg) and from July till December (to 6.8 mg). In the spleen of toads kept in the laboratory the lymphocyte aggregations were diminished in the white pulp while the amount of connective tissues increased both in the white and in the red pulps. Melano-macrophages were more abundant in the red pulp of toads kept in the laboratory than in freshly collected ones. The thymuses of toads kept in the laboratory were decreased in size and depleted of the majority of their cortical lymphocytes. It is suggested that the morphological changes of the yellow-bellied toad lymphoid organs might be the results of stressful laboratory conditions and lower antigenic stimulation in the laboratory than in nature.

Characterization of beta-adrenergic receptors in fish and amphibian lymphoid organs.

Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity beta-adrenergic receptors specific for beta-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian beta1 and beta2-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another beta-AR subtype, or a serotonergic receptor.

Leukocytes of rainbow trout (Oncorhynchus mykiss) pronephros: cell types producing superoxide anion.

Fish pronephric and blood leukocytes yield a chemiluminescent response (CL) when stimulated appropriately. This response reflects the production of highly reactive oxygen derivatives which contribute to oxygen-dependent killing of targets such as pathogens and parasites. From suspensions of pronephric cells of the Rainbow trout, Oncorhynchus mykiss, we have obtained populations enriched for CL-positive cells. Four bands of cells were obtained using continuous gradients generated with 60% Percoll. The leukocytes of band II showed a very strong PMA-induced CL response, the magnitude of which was several times higher than that observed with equivalent numbers of cells form unseparated pronephric cell suspensions. Cells present in other bands were not significantly chemiluminescent. Flow cytometric analysis showed that band II contained large granular cells and small granular cells. Cytochemical analysis showed that this subpopulation was greatly enriched with neutrophils. Many band II cells adhere to glass whereas few band III cells do so. The glass-adherent cells loose their CL potential after a few days in vitro, whereas the nonadherent cells retain their CL responsiveness for at least a week in vitro.

Season-specific thymic architecture in the frog, Rana temporaria: SEM studies.

It is already known that the thymus of the adult common frog, Rana temporaria, undergoes conspicuous annual cyclic changes. Light microscopic (LM) observations are at present confirmed by three-dimensional scanning electron microscopy (SEM) pictures showing the presence of a characteristic cortico-medullary division of summer thymuses and cystic appearance of winter organs. Summer thymuses are larger, much heavier and populated by a significantly higher number of cells than winter organs. The season-specific thymic size, cell content and distribution are reflected by its angioarchitecture. The microcorrosion casts of vascular system of winter thymuses are much smaller and more compact than those of the summer organs in which vessels are arranged at larger spatial intervals. It seems that thymic enlargement following the winter atrophy results from vernal repopulation of the thymic cortex by thymocytes which in consequence induces a reshaping of the existing capillary network.

Rejection of skin allo- and xenografts in the grass frog, Rana temporaria and the edible frog, Rana esculenta.

The fate of orthotopic dorsal skin allo- and xenografts at 22 +!- 2 degrees C in the grass frog, Rana tempororai (Rt) and in the edible frog, R. esculenta (Re) which is an interspecific hybrid of R. ridibunda and R. lessonae. Median survival times (MSTs) in experimental groups Rt in equilibrium Rt, Re in equilibrium Re and Rt comes from Re were 28, 26 and 25 days, respectively. In R. tempororia hosts the significantly shorter viability of xenografts than allogeneic grafts may be caused by the broader spectrum of transplantation antigens of the hybrid donors. In the experimental group Re comes from Rt similar surviva times of sensitizing, second set and third set grafts were observed (24, 22, and 22 days, respectively). The first symptoms of destruction in the grafts of dorsal skin of R. temporaria were visible earlier than in the grafts of dorsal skin of R. esculenta independently of the species of the hosts. Hypotheses which could explain the differences observed are discussed.

Hybrid resistance to the ascites form of the murine sarcoma.

An ascites form (AA) of the methylcholanthrene-induced murine sarcoma of C3H origin was used for present experiments. The AA cells were intraperitoneally injected to syngeneic (C3H, H-2k), allogeneic (DBA, H-2d) or semisyngeneic (C3H x DBA, H-2k/d) mice at a dose 10(7) cells per animal. The DBA mice developed small amount of ascites in the peritoneal cavity followed by rejection of allogeneic tumor cells and recovery. All syngeneic and semisyngeneic animals developed tumor and died. The C3H x DBA hybrids survived significantly longer than C3H mice (mean survival times: 21.9-24.8 and 10.1-11.8 days, respectively). The hybrid mice died with voluminous ascites tumor accompanied by a sponge-like form of tumor cell aggregates dispersed in the peritoneal cavity while the syngeneic mice died with small amount of dense ascites with an accompanying massive solid tumor in the vicinity of the spleen. Therefore in the experimental system used the semisyngeneic mice were found to develop a better response against parental-strain tumor cells than the syngeneic mice. This is an example of the phenomenon referred to as "hybrid resistance" or "allogeneic inhibition".

Rhythms of immunity.

Rhythms of daily activity are found in all vertebrate species, some of them being diurnal (like humans, dogs, pigeons), others--nocturnal (like mice, rats and bats). Some species undergo very pronounced seasonal changes, as they hibernate in the winter or mate only at the specific seasons. The main regulator (a clock and a calendar) for daily and seasonal rhythms is the periodicity of the external light-darkness, reflected by the periodicity of melatonin secretion from the pineal gland, which is inhibited by light and induced during the darkness. In contrast to melatonin which peaks during the night both in diurnal and noctural species, the cyclicity of other hormones and several immune parameters correlates with the pattern of the animal locomotor activity-resting. The immune parameter that peaks at one time of day for a diurnal species peaks about 12 h later for a nocturnal one. Various immune parameters peak at various time points, anticipating an encounter with pathogens during the period of activity while energetically expensive resolution of the immune response during the resting. Daily and seasonal cyclicity of the immune functions are temporally integrated with other physiologic and behavioral processes and all of them are regulated and coordinated with daily and seasonal changes of an external environment by the neuroendocrine homeostatic system.

MHC molecules and lymphocytes: evolutionary perspective.

The classical polymorphic MHC molecules of class I or class II bind peptides derived from the processed cytosolic or endosomal antigens and export them to the cell surface for presentation to the T cell receptors (TcR) of CD8 or CD4 T lymphocytes, respectively. The classical MHC molecules are unstable when peptides are not bound. The MHC-peptide-TcR interactions constitute a molecular basis of thymic selection of the major streams of alpha beta and gamma delta T lymphocytes. The monomorphic MHC class I-like molecules (class Ib) bind peculiar peptides or nonpeptide antigens or can keep proper conformation even without antigenic peptides. They are recognized by the specialized subsets of nonconventional lymphocytes, mainly extrathymic gamma delta T or natural killer (NK) T lymphocytes. The most unorthodox T lymphocytes can see antigens directly without the participation of MHC or MHC-like molecules or can see MHC-like molecules not loaded with peptides. The conventional B2 lymphocytes are indirectly dependent on MHC-peptide-TcR interactions as they can bind the epitopes of native antigens via Ig surface receptors, to be activated they must present the processed antigens via the MHC class II molecules to the Th2 lymphocytes. In contrast, the B1 lymphocytes can be activated directly without cooperation with T cells via MHC molecules. It seems that both MHC molecules and lymphocyte antigen receptors arose by the expansion of Ig-superfamily genes at the early steps of vertebrate phylogeny. The nonconventional T lymphocytes (gamma delta T cells and NK T lymphocytes) and B1 cells which support innate immunity at the body surfaces or cavities as well as the MHC-like molecules might appear earlier, creating a proper microenvironment for development of the conventional T and B2 subsets of lymphocytes.

Studies on transplantation immunity of the yellow-bellied toad Bombina variegata.

Adults and tadpoles of the yellow-bellied toad Bombina variegata reacted in a typically chronic manner to skin allografts and to xenografts from closely related fire-bellied toads B. bombina but they rejected quickly skin xenografts from evolutionary distant anuran species (Bufo and Rana). Adult individuals reacted to allografts slowly not only in the laboratory where their mating was ceased and the weight of lymphoid organs significantly diminished but also in the outdoor enclosure where they bred successfully. Breeding activity in captivity can be induced at any season by Biogonadyl injections. However, any hormonal manipulation (gonadectomy or Biogonadyl treatment) performed during winter/spring on animals housed in the laboratory for several months did not influence their transplantation immunity and the weights of thymuses and spleens. These results lead to conclusion that chronic allograft rejection was not a laboratory artifact caused by a hormonal imbalance but rather reflected a weak donor-host genetic disparity connected with the low MHC polymorphism of Bombina species.

Morphine modulation of thioglycollate-elicited peritoneal inflammation in the goldfish, Carassius auratus.

Intraperitoneal (i.p.) injection of adult goldfish with 3% thioglycollate (TG) induces an acute peritoneal inflammation connected with a massive influx of inflammatory leukocytes mainly of the head kidney origin. The number of peritoneal exudate cells retrieved on day 2 of the inflammatory response is significantly lower in fish injected with TG and morphine (20 mg/kg b.w.) than in animals injected with TG only. The morphine effect was totally antagonized in fish injected 20 min earlier with naltrexone (1 mg/kg), a well-known blocker of opioid receptors. Light microscopy of the head kidney Epon sections revealed that basophilic granulocytes are common in the control PBS-injected fish and even more frequent in fish injected with morphine only. In sharp contrast basophils are very rare in the head kidneys from animals with the TG-induced peritoneal inflammation, while they are more numerous in fish injected with TG and morphine. Supposedly, the basophilic granulocytes might be involved in the inhibitory effects of morphine on the acute inflammation in goldfish. An involvement of the head kidney in the morphine modulation of the inflammatory response is strongly supported by the detection of opioid receptors in the head kidney cells suspension.

Ultrastructure of the APUD-like cells of the frog thymus gland.

Within the thymus gland of the European common frog, Rana temporaria, cells with endocrine- like appearance have been found. At the ultrastructural level the most characteristic feature of their cytoplasm is the presence of secretory granules. Some cells possess irregular electron lucent granules with an eccentrically located dense core while others possess smaller electron dense granules. The cytoplasm contains also cisterns of rough endoplasmic reticulum, free ribosomes, and small mitochondria. The cells possess irregular nuclei with the pronounced nucleoli. These endocrine-like cells are connected by desmosomes with neighbouring non-granulated epithelial cells. Ultrastructural features of the cells described here resemble those seen in polypeptide hormone-secreting cells belonging to the family of cells of the APUD (Amine Precursor Uptake and Decarboxylation) series.

Interdigitating cells in the thymus of the frog, Rana temporaria.

Interdigitating cells (IDC) of the thymic medulla of the frog, Rana temporaria, collected in the summer, were examined by electron microscopy. The most characteristic cytological features of IDC are voluminous electron-lucent cytoplasm and widespread interdigitations and invaginations of the cell membrane. IDC possess an excentrically located nucleus with pronounced nucleoli and a thin rim of a dense chromatine as well as a perinuclear area with characteristic tubulo-vesicular complex. In our material Birbeck granules were absent. Some IDC contain phagocytized material. A few transitional forms between monocytes and IDC were observed. On the basis of these observations it is highly probable that the amphibian IDC belong to the mononuclear phagocyte system.

Cytoarchitecture of the thymus gland of the adult frog (Rana temporaria).

Like in other jawed vertebrates, the frog thymus consists of lymphoid cells within epithelial framework and characteristic myoid cells. Mammalian type Hassall's corpuscles are absent, but degenerative cells form so-called unicellular Hassall's bodies. Conspicuous secretory cells, secretory and degeneration cysts as well as phagocytic cells, granulocytes and plasma cells can be observed in the frog thymus. Interdigitating-like cells and some characteristic features of thymic vasculature (i.e. the existence of blood-thymus endothelial are preliminary described in the frog thymus.

Effects of hydrocortisone on the lymphoid organs of Rana temporaria.

In vivo and in vitro effects of hydrocortisone hemisuccinate (HC) on the lymphoid organs were studied in the European common frog Rana temporaria. Single HC injection at the dose of 0.5 mg/g body weight induced rapid but fully reversible changes in thymuses and jugular bodies while its effect on spleens was negligible. In vitro studies confirmed HC-sensitivity of thymuses and jugular bodies and relative HC-resistance of spleens of this species. In vitro exposition of thymic and jugular body cell suspensions to 0.25-1.00 mg/ml HC solution revealed a dose-dependent and time-dependent impairment of sample activities associated with massive cell death as judged by MTT reduction assay and Trypan blue uptake, respectively. Evolutionary implications of these results are discussed.

Argentaffin mast cells in the thymus of the frog.

Mast cells are common in the thymic parenchyma of the European common frog, Rana temporaria. They are stained meta chromatically with toluidine blue and the majority of them are impregnated with silver during the argentaffin reaction. The latter phenomenon indicate that these cells store serotonin. At the ultrastructural level, mast cells contain specific granules with electron-dense and electron-lucent parts. The silver grains are located exclusively over the electron-lucent part of the mast cell granules indicating that serotonin is stored just in this compartment.

Light and electron-microscopic observations on the thymic capillaries and postcapillary venules in Rana temporaria L.

The aim of this study was to examine thymic capillaries and capillary venules in a common frog, Rana temporaria, using the light (LM) and transmission electron microscopy (TEM) as well as scanning electron microscopy (SEM) of vascular corrosion casts. The endothelial cells of capillaries and postcapillary venules show in frog thymus mostly flat morphology. However, some postcapillary venules are lined with tall endothelial cells which are characteristic for the thymus-dependent areas of mammalian secondary lymphatic organs (high endothelial venules, HEV). Such endothelial cells occurring in frog thymus are ultrastructurally characterized by increased amount of cytoplasmic organelles, mainly rough endoplasmic reticulum, ribosomes and electron dense lysosome-like granules, as well as by deep intercellular clefts. In HEV-like vessels, lymphocytes were observed to adhere to the endothelial surface, migrate through the endothelium, or lay beneath the endothelial basement membrane in the perivascular space. These findings were corroborated by SEM of corrosion casts which revealed in some postcapillary venules or their segments the presence of irregular casts surfaces with numerous depressions and protrusions reflecting the cobblestone appearance of the tall endothelial lining. Such venules are fed directly by capillaries and begin with abrupt increase in the vessel diameter, accompanied by the appearance of deep endothelial imprints. Our results demonstrate in the frog thymus the presence of specialized, HEV-like segments of postcapillary venules, suggesting that besides its function as the central lymphoid organ, frog thymus may also function as a peripheral one.